It is now generally accepted that membrane proteins may undergo lateral diffusion in the plan of the cell membrane, and such lateral mobility may play important roles in cellular functions and cell-cell interactions. There are three important issues concerning protein diffusion in cell membranes: (1) Exactly how fast may membrane proteins diffuse in the membrane? (2) Are membrane proteins capable of lateral diffusion in vivo? (3) Is their diffusional mobilty restricted in mature cells? These issues remained unresolved for two reasons: First, themost popular technique used in meausring protein diffusion in cell membrane, namely, the fluorescence photobleaching recovery method, has been controversial because of possible photobleaching-induced artifacts. Secondly, most of the studies on protein diffusion were carried out in tissue culture or embryonic cell preparations. In the present project, we propose to compare and evaluate three existing biophysical methods (fluorescence photobleaching recovery, post-field relaxation, and asymmetric inactivation recovery) by measuring the lateral diffusion of the same membrane proteins (lectin and acetylcholine receptors) in the membrane of cultured Xenopus muscle cell. We also propose to compare the diffusion rates of the same membrane protein (acetylcholine receptor) in the embryonic and mature stages of the muscle cell, using in vivo preparations of Xenopus tadpole myotomes and mature denervated muscle fibers of the frog. Results from these studies will provide useful information for a resolution of the above-mentioned issues concerning protein diffusion in cell membranes, and for further understanding of the role of lateral mobility of membrane proteins in their biological functions.